Mutations in the T-box transcription factor TBX5 cause Holt-Oram syndrome, an autosomal dominant human disorder characterized by radial hand and arm deformities and pleiotropic cardiac malformations. We hypothesize that the consequences of Holt-Oram mutations in the TBX5 transcription factor cause multiple abnormalities in DNA binding, dimerization, and transcriptional activation or repression domain functions, and that these properties can be measured and used to generate models of TBX5 structure and function. In order to pursue this hypothesis we have identified a TBX5 binding site that is bound by monomeric and dimeric TBX5 in gel shift assay. We have constructed a test cell for measuring the DNA binding kinetics of TBX5 in real time by surface plasmon resonance monitoring and used it to demonstrate that the R237Q mutation of this transcription factor has dominant negative effects on DNA binding by wild-type TBX5. We have mapped activation domains in yeast and demonstrated complex interactions between the 1-box and activation domains in the amino and carboxy terminus of TBX5. We show that the alpha and beta isoforms of this protein have distinguishable domain structures in yeast and that they have different effects in transient transfection assays in cardiac myocytes. Our goals in this application are to define mechanisms by which TBX5 mutations result in dysfunction of TBX5 as a transcription factor and to identify additional mutations in the TBX5 transcription factor associated with cardiac disease. Our investigations will enhance current insights into the genetic mechanisms responsible for cardiac malformations arising from TBX5 mutations specifically and may ultimately provide a general mechanistic model for understanding interrelationships between transcription factor mutations, transcription factor function, and abnormal cardiovascular development.